Inhibition of constitutive protein secretion from lactating mouse mammary epithelial cells by FIL (feedback inhibitor of lactation), a secreted milk protein

1993 ◽  
Vol 106 (2) ◽  
pp. 641-648 ◽  
Author(s):  
M.E. Rennison ◽  
M. Kerr ◽  
C.V. Addey ◽  
S.E. Handel ◽  
M.D. Turner ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Early Stage ◽  
Regulatory Protein ◽  
Brefeldin A ◽  
Mouse Mammary Gland ◽  
Mammary Epithelial ◽  
Feedback Inhibitor

The effect of a protein feedback inhibitor of lactation (FIL) on casein synthesis and secretion was examined using isolated acini from lactating mouse mammary gland. As previously found, FIL partially inhibited protein synthesis but produced an additional inhibition of constitutive casein secretion. The inhibition of synthesis and secretion showed similar dose-dependency and the inhibition was fully reversible. Constitutive secretion of pre-formed protein was inhibited by FIL in a pulse-chase protocol, indicating that the inhibitor regulated protein secretion by reducing protein movement through the secretory pathway independently of any initial inhibition of synthesis. Regulated exocytosis was not inhibited since casein release due to elevation of cytosolic Ca2+ concentration by the ionophore ionomycin was unaffected. Brefeldin A, which is known to block ER-to-Golgi transport, also inhibited both protein synthesis and secretion in mammary cells. The action of FIL on synthesis and secretion and previously described actions on casein degradation would be consistent with a block at an early stage in the secretory pathway. In support of this idea FIL treatment was found to result in vesiculation and swelling of the endoplasmic reticulum. These data provide evidence for a novel control of a constitutive secretory pathway by a physiological extracellular regulatory protein.

Investigation of the role of microtubules in protein secretion from lactating mouse mammary epithelial cells

1992 ◽  
Vol 102 (2) ◽  
pp. 239-247 ◽  
Author(s):  
M.E. Rennison ◽  
S.E. Handel ◽  
C.J. Wilde ◽  
R.D. Burgoyne
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Early Stage ◽  
Major Effect ◽  
Vesicle Transport ◽  
Mammary Epithelial ◽  
Mammary Cells

Disruption of microtubules has been shown to reduce protein secretion from lactating mammary epithelial cells. To investigate the involvement of microtubules in the secretory pathway in these cells we have examined the effect of nocodazole on protein secretion from mammary epithelial cells derived from the lactating mouse. Mouse mammary cells have extensive microtubule networks and 85% of their tubulin was in a polymeric form. Treatment with 1 micrograms/ml nocodazole converted most of the tubulin into a soluble form. In a continuous labelling protocol it was found that nocodazole did not interfere with protein synthesis but over a 5 h period secretion was markedly inhibited. To determine whether the inhibition was at the level of early or late stages of the secretory pathway mammary cells were pulse-labelled for 1 h to label protein throughout the secretory pathway before nocodazole treatment. When secretion was subsequently assayed it was found to be slower and only partially inhibited. These findings suggest that the major effect of nocodazole is on an early stage of the secretory pathway and that microtubules normally facilitate vesicle transport to the plasma membrane. An involvement of microtubules in vesicle transport to the plasma membrane is consistent with an observed accumulation of casein vesicles in nocodazole-treated cells. Exocytosis stimulated by the calcium ionophore ionomycin was unaffected by nocodazole treatment. We conclude from these results that the major effect of nocodazole is at an early stage of the secretory pathway, one possible target being casein vesicle biogenesis in the trans-Golgi network.

Differential effect of brefeldin A on phosphorylation of the caseins in lactating mouse mammary epithelial cells

1993 ◽  
Vol 106 (4) ◽  
pp. 1221-1226 ◽  
Author(s):  
M.D. Turner ◽  
S.E. Handel ◽  
C.J. Wilde ◽  
R.D. Burgoyne
Keyword(s):  
Differential Effect ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Milk Proteins ◽  
Brefeldin A ◽  
Mouse Mammary Gland ◽  
Mammary Epithelial ◽  
Secretory Proteins ◽  
Mammary Cells ◽  
Regulated Secretory Pathway

The major milk proteins, the caseins, contain multiple phosphorylation sites. Phosphorylation of the caseins is necessary to allow Ca2+ binding and aggregation of the caseins to form micelles. We have followed the phosphorylation of the caseins in isolated acini from lactating mouse mammary gland. Incubation of mammary cells with [32P]orthophosphate revealed that phosphorylation of newly synthesised caseins was complete within 20 minutes of synthesis. Extensive secretion of alpha-, beta- and gamma-caseins occurred over a 2 hour period. Activation of the regulated secretory pathway using ionomycin over the last hour resulted in a preferential increase in secretion of alpha- and gamma-caseins. Brefeldin A (BFA) inhibited protein secretion and synthesis in mammary cells in prolonged incubations. An examination of short-term treatments with BFA on 32P incorporation into the caseins revealed a differential effect of BFA in which the drug inhibited phosphorylation of beta- and gamma- but not alpha-caseins. These results suggest that phosphorylation of alpha-casein normally occurs in Golgi cisternae whereas that of beta- and gamma-caseins occurs in the trans-Golgi network. Phosphorylation of specific secretory proteins may, therefore, occur in different Golgi compartments.

scholarly journals Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells

1992 ◽  
Vol 117 (2) ◽  
pp. 269-278 ◽  
Author(s):  
MD Turner ◽  
ME Rennison ◽  
SE Handel ◽  
CJ Wilde ◽  
RD Burgoyne
Keyword(s):  
Epithelial Cells ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Isolated Cells ◽  
Pulse Label ◽  
Constitutive Secretion

Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine into protein linearly for at least 5 h with no discernible lag period. In contrast, protein secretion was only detectable after a lag of approximately 1 h, consistent with exocytotic secretion of proteins immediately after passage through the secretory pathway and package into secretory vesicles. The extent of protein secretion was unaffected by the phorbol ester PMA, 8-bromo-cAMP, or 8-bromo-cGMP but was doubled by the Ca2+ ionophore ionomycin. In a pulse-label protocol in which proteins were prelabeled for 1 h before a chase period, constitutive secretion was unaffected by depletion of cytosolic Ca2+ but ionomycin was found to give a twofold stimulation of the secretion of presynthesized protein in a Ca(2+)-dependent manner. Ionomycin was still able to stimulate protein secretion after constitutive secretion had terminated. These results suggest that lactating mammary cells possess both a Ca(2+)-independent constitutive pathway and a Ca(2+)-activated regulatory pathway for protein secretion. The same proteins were secreted by both pathways. No ultrastructural evidence for apocrine secretion was seen in response to ionomycin and so it appears that regulated casein release involves exocytosis. Ionomycin was unlikely to be acting by disassembling the cortical actin network since cytochalasin D did not mimic its effects on secretion. The regulated pathway may be controlled by Ca2+ acting at a late step such as exocytotic membrane fusion.

scholarly journals Brefeldin A inhibits transport of the glycophorin-binding protein from Plasmodium falciparum into the host erythrocyte

1994 ◽  
Vol 300 (3) ◽  
pp. 821-826 ◽  
Author(s):  
J Benting ◽  
D Mattei ◽  
K Lingelbach
Keyword(s):  
Plasmodium Falciparum ◽  
Golgi Apparatus ◽  
Host Cell ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Binding Protein ◽  
Brefeldin A ◽  
Cell Cytoplasm ◽  
Parasite Cell ◽  
Host Cell Cytoplasm

Plasmodium falciparum, a protozoan parasite of the human erythrocyte, causes the most severe form of malaria. During its intraerythrocytic development, the parasite synthesizes proteins which are exported into the host cell. The compartments involved in the secretory pathway of P. falciparum are still poorly characterized. A Golgi apparatus has not been identified, owing to the lack of specific protein markers and Golgi-specific post-translational modifications in the parasite. The fungal metabolite brefeldin A (BFA) is known to inhibit protein secretion in higher eukaryotes by disrupting the integrity of the Golgi apparatus. We have used the parasite-encoded glycophorin-binding protein (GBP), a soluble protein found in the host cell cytoplasm, as a marker to investigate the effects of BFA on protein secretion in the intracellular parasite. In the presence of BFA, GBP was not transported into the erythrocyte, but remained inside the parasite cell. The effect caused by BFA was reversible, and the protein could be chased into the host cell cytoplasm within 30 min. Transport of GBP from the BFA-sensitive site into the host cell did not require protein synthesis. Similar observations were made when infected erythrocytes were incubated at 15 degrees C. Incubation at 20 degrees C resulted in a reduction rather than a complete block of protein export. The relevance of our findings to the identification of compartments involved in protein secretion from the parasite cell is discussed.

Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments

1995 ◽  
Vol 108 (2) ◽  
pp. 519-527 ◽  
Author(s):  
P.L. Jones ◽  
N. Boudreau ◽  
C.A. Myers ◽  
H.P. Erickson ◽  
M.J. Bissell
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Protein Synthesis ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Active Regions ◽  
Mammary Epithelial ◽  
Dependent Manner ◽  
Beta Casein ◽  
Casein Protein

The physiological role of tenascin in vivo has remained obscure. Although tenascin is regulated in a stage and tissue-dependent manner, knock-out mice appear normal. When tenascin expression was examined in the normal adult mouse mammary gland, little or none was present during lactation, when epithelial cells actively synthesize and secrete milk proteins in an extracellular matrix/lactogenic hormone-dependent manner. In contrast, tenascin was prominently expressed during involution, a stage characterized by the degradation of the extracellular matrix and the subsequent loss of milk production. Studies with mammary cell lines indicated that tenascin expression was high on plastic, but was suppressed in the presence of the laminin-rich, Engelbreth-Holm-Swarm (EHS) tumour biomatrix. When exogenous tenascin was added together with EHS to mammary epithelial cells, beta-casein protein synthesis and steady-state mRNA levels were inhibited in a concentration-dependent manner. Moreover, this inhibition by tenascin could be segregated from its effects on cell morphology. Using two beta-casein promoter constructs attached to the chloramphenicol acetyltransferase reporter gene we showed that tenascin selectively suppressed extracellular matrix/prolactin-dependent transcription of the beta-casein gene in three-dimensional cultures. Finally, we mapped the active regions within the fibronectin type III repeat region of the tenascin molecule that are capable of inhibiting beta-casein protein synthesis. Our data are consistent with a model where both the loss of a laminin-rich basement membrane by extracellular matrix-degrading enzymes and the induction of tenascin contribute to the loss of tissue-specific gene expression and thus the involuting process.

scholarly journals Exocytosis from permeabilized lactating mouse mammary epithelial cells. Stimulation by Ca2+ and phorbol ester, but inhibition of regulated exocytosis by guanosine 5′-[γ-thio]triphosphate

1992 ◽  
Vol 286 (1) ◽  
pp. 13-15 ◽  
Author(s):  
M D Turner ◽  
C J Wilde ◽  
R D Burgoyne
Keyword(s):  
Epithelial Cells ◽  
Protein Secretion ◽  
Phorbol Ester ◽  
Mammary Epithelial Cells ◽  
Post Partum ◽  
Mammary Epithelial ◽  
Independent Manner ◽  
Permeabilized Cells ◽  
A Site ◽  
Golgi Network

Lactating mouse mammary epithelial cells secrete large amounts of milk protein via constitutive or regulated exocytotic pathways. Secretion through both pathways was quantified by assaying the release of [35S]methionine-labelled trichloroacetic acid-precipitable proteins from digitonin-permeabilized secretory acini isolated from mammary glands of 10-day-post-partum lactating mice. Protein secretion from the isolated permeabilized cells was either Ca(2+)-dependent (regulated) or Ca(2+)-independent (constitutive). In both cases there was a requirement for ATP. Addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) caused a marked increase in the percentage protein secretion from the cells in a Ca(2+)-independent manner. However, the non-hydrolysable GTP analogue guanosine 5′-[gamma-thio]triphosphate (GTP[S]) caused a partial inhibition of Ca(2+)-dependent exocytosis, while having no significant effect on Ca(2+)-independent exocytosis. Thus the GTP[S] is exerting its effect on the regulated pathway at a site subsequent to protein sorting and packaging into secretory vesicles at the trans-Golgi network.

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